The propensity for blood to clot too rapidly is an important prognosticator for the development of, progression of, and recovery from a number of serious pathological conditions whose pathogeneses either arise directly from or are modulated by the blood clotting process. These diseases include heart attack, stroke, coronary artery disease, deep vein thrombosis, and pulmonary embolism, among others. Of these diseases, coronary artery disease is the leading cause of mortality in the United States. Furthermore, certain clinical conditions may predispose patients to undergo adverse clotting phenomena, such as vascular disease, surgery, trauma, malignancy, prosthetic vascular devices, general anesthesia, pregnancy, use of oral contraceptives, systemic lupus erythematosus, and infection. Often, patients with acute conditions suspected of resulting from clotting abnormalities appear in the emergency room. Means for rapidly detecting in a blood sample the propensity for clot formation may help rule in or rule out thrombotic events and coagulopathies and improve the delivery of emergency health care to those in need, while also offering early identification of patients who may progress to potentially lethal clotting pathology. It has been estimated that 80% of all deaths are associated with a disease in which abnormal clotting phenomena occur, principally of blood that clots too quickly.
Blood clotting is a complex process involving multiple initiators, cascades of activators, enzymes, and modulators which ultimately lead to the formation of fibrin, which polymerizes into an insoluble clot. Classically, the propensity for blood to clot is measured manually or automatedly by measuring the time taken for a sample of plasma or blood to form insoluble fibrin strands or a clot. Clot formation may be detected visually, by observing the formation of fibrin strands, or by automated means such as by changes in viscosity or by photo-optical detection of the clot in plasma-based assays. The measurement of clotting time may be made immediately on freshly drawn blood without the need for addition of anticoagulants, or may be made on blood containing a calcium-binding anticoagulant such as citrate by adding a calcium salt to reverse the anticoagulant effect; this latter determination is referred to as recalcification time. Determination of the coagulation time has been most commonly used for the diagnosis of diseases such as hemophilia, von Willebrand's disease, Christmas disease and hepatic diseases, wherein abnormally prolonged clotting times are usually diagnostic. Typical methods for the measurement of blood coagulation time which have been conventionally employed include those relying on the measurement of prothrombin time (PT), the measurement of activated partial thromboplastin time (APTT), the measurement of thrombin time, as well as the fibrinogen level test. Detection of a thrombotic event also may be performed by measuring the level of soluble fibrin or fibrin degradation products in circulation. Although many impending serious conditions involving abnormal blood coagulability might be detectable prior to the occurrence of acute, lethal or near-lethal events, altered blood coagulability measurement methods are not presently sensitive enough to be diagnostically valuable in identifying all but the most abnormal coagulation samples.
The PT and APTT tests are not sensitive enough to be used to detect hypercoagulable states, and are generally used to detect conditions with prolonged clotting times. These tests are usually performed on plasma, which does not contain activated platelets and monocytes, both of which may contribute significantly to altered coagulation states. Furthermore, these tests utilize reagents added to the sample which are procoagulants themselves and reduce the clotting time of plasma from about six minutes to values of about 12 seconds, and 38 seconds, for PT and APTT, respectively. By excluding the influence of the cellular components of whole blood, such as monocytes, these popular measurement methods for clotting time using plasma as described above do not fully provide maximum predictive and diagnostic value for thrombotic events that may be modulated by the cellular components of blood. Furthermore, the monitoring of anticoagulant therapies such as heparin and warfarin would be improved if the coagulability of whole blood, rather than plasma alone, were measured. The presence of therapeutically-administered anticoagulants modulates coagulability through cellular as well as soluble (plasma) blood constituents.
One important initiator and modulator of the blood clotting process is a procoagulant enzyme called tissue factor present which may be present in and on the surfaces of both endothelial cells, which line the vasculature, and monocytes, which circulate in blood. Increased expression of tissue factor by these cell types has been linked to many thrombotic disorders and pathologic states. The ability of monocytes to generate tissue factor is well known. However, the majority of monocyte membrane-associated tissue factor is not in an enzymatically active form that can initiate clotting; in order to become active, tissue factor must form an active complex with another of the clotting factors, Factor VII or its activated form, Factor VIIa. The Tissue Factor:Factor VIIa complex may then activate zymogens Factor IX and Factor X to their enzymatically active forms, Factors IXa and Xa. Factor Xa combines with prothrombin to yield the prothrombinase complex (active procoagulant), yielding thrombin which cleaves fibrinogen, finally yielding fibrin which forms the clot. The level on blood monocytes of the inactive, latent form of tissue factor, and its proclivity to become activated and eventually to form the prothrombinase complex, is a diagnostically useful parameter for identifying patients at risk of undergoing thrombotic events. Tissue factor has been found on circulating monocytes from patients with cancer, infections, and thrombotic disorders such as heart attack and stroke.
Methods for the direct measurement of tissue factor level have been described. In addition to immunoassay procedures, such as that described in U.S. Pat. No. 5,403,716, the exposure of whole blood to endotoxin, as described in U.S. Pat. No. 4,814,247 and as described by Spillert and Lazaro, 1993, J. Nat. Med. Assoc. 85:611-616, provides within several hours an assessment of tissue factor levels. In this invention, the modified recalcification time is measured in a blood sample exposed to endotoxin for a period of time adequate for active tissue factor to be generated by monocytes in the sample.
Kaneko et al. (1994, Br. J. Haem. 87:87-93) determined that a monocyte leukemia cell line (U937 cells) when incubated with mercuric ion or silver ion in tissue culture had increased generation of tissue factor-dependent procoagulant activitv. This increased procoagulant activity was postulated to be a result of changes of cell surface phosphatidylserine and other membrane changes.
The invention described herein offers a rapid method for assessment of the thrombotic activity of blood by measuring blood clotting time after exposure to certain heavy metal ions such as those of mercury and silver.